| 建立NHEJ修复定量体系并检测拓扑异构酶抑制剂依托泊苷对NHEJ的影响 |
| 投稿时间：2015-01-15 修订日期：2015-01-26 点此下载全文 |
| 引用本文：李莉萍,吴晓丹,孙有湘,陈利俊.建立NHEJ修复定量体系并检测拓扑异构酶抑制剂依托泊苷对NHEJ的影响[J].医学研究杂志,2015,44(8):26-32 |
| DOI：
10.11969/j.issn.1673-548X.2015.08.009 |
| 摘要点击次数: 1314 |
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| 基金项目:国家自然科学基金资助项目(31271436);广东省科技计划项目(2010B060900109);广东省自然科学基金资助项目(S2012010008225);广东省中医药局课题(1050051) |
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| 中文摘要:目的 建立含pI-SCEⅠ酶切位点的总的非同源末端连接(total non-homologous end joining,Total-NHEJ) 和非经典末端连接(alternative end joining,A-EJ)修复底物的细胞株,设置特定的流式细胞术参数,定量检测依托泊苷(etoposide,VP-16)对非同源末端连接(Non-homologous end joining,NHEJ)修复的影响。 方法 本实验通过脂质体转染含修复底物的质粒,嘌呤霉素筛选,构建起NHEJ修复系统的稳定细胞株;运用流式细胞术和PCR技术对所构建的细胞株的基因组DNA进行分析与鉴定;运用细胞免疫荧光及基因组DNA琼脂糖凝胶电泳检测VP-16诱导的DNA双链断裂(DNA double strand break,DSB)损伤,运用流式细胞术定量检测VP-16对NHEJ修复的影响。 结果 在所筛选的细胞中的各得到2株阳性细胞株Total-NHEJ和2株阳性细胞株A-EJ。VP-16作用浓度和时间的增加,Total-NHEJ 和A-EJ修复效率增加。 结论 VP-16诱导DNA损伤的同时,促进总NHEJ修复和A-EJ修复,修复呈现剂量和时间依赖性,但随着浓度和作用时间的增加,VP-16致DSB的损伤能力超过修复能力。 |
| 中文关键词:稳定转染 非同源末端连接 非经典末端连接 依托泊苷 |
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| Establishing NHEJ Repair Quantitative Detection System and Detecting the Roles of Topoisomerase Inhibitors Etoposide on NHEJ |
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| Abstract:Objective To establish two cell lines containing restriction site of pI-SCEⅠ and separately reporters of total-NHEJ(total non-homologous end joining)and alternative end joining(A-EJ),set specific parameters of flow cytometry to detect the role of etoposide(VP-16) played in total-NHEJ and A-EJ quantificationally. Methods In this study,we transfer plasmids containing repair substrates using liposomal,establish stable cell lines of NHEJ repair system by subsequent puromycin screening,identified genomic DNA of the two cell lines by flow cytometry and PCR,detected VP-16-induced DSB damage by immunofluorescence and genomic DNA agarose gel electrophoresis,detected the effect of VP-16 on NHEJ repair useing flow cytometry quantificationally. Results Among the clones,2 clones belongs to pI-SCEⅠ+ Total-NHEJ cells, and 2 clones belongs to pI-SCEⅠ+ A-EJ cells.With the increase of VP-16 concentration and action time, NHEJ repair effects increased. Conclusion The results showed that VP-16 induced DNA damage at the same time, promoted the total NHEJ repair and A-EJ repair.The repair is dose and time dependent, but with the increase of concentration and action time, the capacity VP-16 cause to DSB damage exceeds its repair effects. |
| keywords:Stable transfection Non-homologous end joining Alternative end joining Etoposide |
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